Arginine deiminase derived from Mycoplasma arthritidis and polymer conjugates containing the same

ABSTRACT

A purified arginine deiminase (ADI) obtained from Mycoplasma arthritidis having the amino acid sequence of SEQ ID NO:2 as well as an isolated nucleic acid molecule containing a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:1 are disclosed. Other aspects of the invention include an expression vector, a cloned gene for expressing the Mycoplasma arthritidis derived ADI, (recombinant) host cells useful in expressing the ADI of the present invention and substantially non-antigenic polymer conjugates containing the ADI of the present invention as well as methods of treating arginine deiminase susceptible conditions in mammals. The arginine deiminase-polymer conjugates have high levels of retained enzyme activity and relatively long circulating lives.

The present application is a divisional of U.S. Ser. No. 08/792,283,filed on Jan. 31, 1997, now U.S. Pat. No. 5,804,183, the contents ofwhich is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

The present invention is directed to a novel arginine deiminase andlong-acting arginine deiminase-containing compositions. In particularthe invention is directed to substantially non-antigenic polymerconjugates containing arginine deiminase derived from Mycoplasmaarthritidis which demonstrate high levels of retained enzyme activity.

Conjugating biologically-active proteins or enzymes to polymers has beensuggested to improve one or more of the properties of circulating life,water solubility or antigenicity in vivo. For example, some of theinitial concepts of coupling peptides or polypeptides to polyethyleneglycol (PEG) and similar water-soluble polymers are disclosed in U.S.Pat. No. 4,179,337, the disclosure of which is incorporated herein byreference. Conjugates are formed by reacting a biologically activematerial with a several fold molar excess of a polymer which has beenmodified to contain a terminal linking group. Insulin and hemoglobinwere among the first therapeutic agents conjugated. These relativelylarge polypeptides contain several free ε-amino attachment sites.Several polymers could be attached without significant loss of biologicactivity. The conjugation process, however, is not withoutcomplications. Excessive polymer conjugation or reactions using molarexcesses of polymers beyond certain ratios can result in the formationof inactive conjugates or conjugates having insufficient activity.Problems often result when the active site (i.e. where groups associatedwith bioactivity are found) on the protein or enzyme becomes blocked asa result of the covalent polymer attachment. This problem can bedifficult to avoid because the polymer and protein or enzyme aretypically joined in solution-based reactions. Pre-blocking the activesites with reversible materials such as pyridoxal phosphate has beensuggested, but the results have been inconsistent. The problems areparticularly acute with relatively lower molecular weight proteins andpeptides. These bioactive materials often have few attachment sites notassociated with bioactivity.

Arginine deiminase (ADI) is one type of enzyme which could benefit fromimproved polymer conjugation techniques. ADI is an enzyme thathydrolyzes arginine and the depletion of arginine has been postulated tohave a killing effect on certain tumors. In particular, the enzymehydrolyzes the guanidino group of L-arginine into L-citrulline andammonia. The ADI enzyme is also known as arginine dihydrolase, argininedesiminase or guanidinodesiminase. Although ADI has been obtained fromother types of mycoplasma strains as well as other microorganism sourcessuch as pseudomonas and streptococcus, there is variability in theenzyme obtained. In particular, each host strain produces an enzymehaving a different number of lysines as well as variations in the activesite to produce an enzyme having a different primary sequence, andnumber and location of lysines.

Several polymer-arginine deiminase conjugates have previously beensuggested. See, for example, Jpn. J. Cancer Res. 84, 1195-1200, November1993 which describes inter alia arginine deiminase from Mycoplasmaarginini conjugated with methoxy-polyethyleneglycol-4,6-dichloro-1,3,5-triazine. The previous argininedeiminase-polymer conjugates prepared to date, however, have been deemedto be unacceptable. One of the chief drawbacks has been that the levelof retained arginine deiminase activity provided by highly modifiedconjugates has been too low in growth inhibition studies. It has beenpostulated that certain lysine attachment points on the enzyme areintimately connected with the enzyme active site. Therefore, the highlymodified conjugates which demonstrate high levels of retained activitywere not possible at reasonable expenditures of time and resources.

The present invention addresses these shortcomings.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a novel purified argininedeiminase subunit (hereinafter ADI) obtained from Mycoplasma arthritidishaving the amino acid sequence of SEQ ID NO:2. In this regard, theinvention includes an isolated nucleic acid molecule containing anucleotide sequence encoding ADI comprising the amino acid sequence setforth in the Figures and SEQ ID NO:1 and SEQ ID NO:2 as well as nucleicacid molecules complementary thereto.

Other aspects of the invention include an expression vector containing acloned gene for Mycoplasma arthritidis derived ADI, (recombinant) hostcells useful in expressing the ADI of the present invention andsubstantially non-antigenic polymer conjugates containing the ADI of thepresent invention. Still further aspects of the invention include aprocess for preparing the purified ADI, a process for preparing theaforementioned arginine deiminase-containing substantially non-antigenicpolymer-based compositions as well as methods of treating argininedeiminase susceptible conditions in mammals. In this aspect, thetreatment methods include administering an effective amount of thecompositions described herein, preferably as part of a polymerconjugate, to mammals in need of such therapy.

The substantially non-antigenic polymer is preferably a polyalkyleneoxide such as a polyethylene glycol having a molecular weight of fromabout 600 to about 60,000 and preferably having a molecular weight ofabout 12,000. Other substantially non-antigenic polymers can also beused. The substantially non-antigenic polymer is preferably covalentlyconjugated to the ADI via a urethane or similarly hydrolysis resistantlinkage in one preferred embodiment. The arginine deiminase-polymerconjugate containing compositions can be included as part of apharmaceutically-acceptable solution.

The ADI obtained from M. arthritidis in accordance with the presentinvention differs from that previously reported in J. Biol. Chem. Vol.253 No. 17 pp 6016-6020 (1978) and J. Biol. Chem. Vol. 252 No. 8 pp2615-2620 (1977). Although both ADI's were obtained from the sameAmerican Type Culture Collection ("ATCC") 14152 strain, the ADIdescribed herein has only two cysteines per subunit sequence. Thepreviously reported protein included eighteen cysteines per subunit.Secondly, the ADI of the present invention has a pI of about 5.25whereas the previously reported ADI obtained from M. arthritidis had apI of 7.0. In addition, the previous work reported above also predictedthat the ADI obtained from M. arthritidis would have fewer lysines persubunit than the ADI from M. arginini. The present inventors, however,have surprisingly discovered that the opposite was the case. A stillfurther point of difference is the fact that the N-terminal residue ofthe ADI of the present invention is serine(following post-translationalremoval of methionine), while the previously reported M. arthritidis ADIreported alanine as the N-terminal residue. These differences aredramatic and suggest that there is heterogeneity in the ADI obtainedfrom obtained from M. arthritidis. A still further possibility is thatthere is more than one gene for ADI activity associated with M.arthritidis.

The arginine deiminase-polymer conjugates of the present invention alsoafford advantages over those of the prior art. For example, the ADI ofthe present invention includes several more modifiable lysine positionsthan prior art ADI, without resorting to the preparation of mutantlysine variants. This allows for substantially more polymer strands tobe covalently attached to alternate surface locations without losingtumor cell growth inhibition activity. In addition, the thus formedconjugates have a substantially longer in vivo circulating life thanconjugates having similar levels of retained activity prepared accordingto the prior art.

The term "arginine deiminase susceptible condition" shall be understoodto include all disease states, such as tumor growths, cancers, orrelated conditions, which benefit therapeutically from exogenousarginine deiminase administration. Details concerning such conditionsare provided below in Section 4.

For a better understanding of the present invention, reference is madeto the following description and its scope will be pointed out in theappended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the complete DNA sequence encoding ADI cloned from M.arthritidis ATCC 14152 (1230 bases). The underlined G in the5-tryptophan codons were changed from A to G by site directedmutagenesis.

FIG. 2 illustrates the translation product (410 amino acids) of thearginine deiminases subunit from M. arthritidis ATCC 14152.

FIG. 3 illustrates the amino acid sequence alignment of various argininedeiminases; line a: M. arthritidis ATCC 14152 (SEQ ID NO:2); line b: M.arginini LBIF (SEQ ID NO:7), see Ohno et al. Infection and Immunity 58:November 1990, pp 3788-3795; line c: M. hominis PG21 (SEQ ID NO:8); lined: M. orale FERM BP-1970 (SEQ ID NO:9). M. hominis and M. orale obtainedaccording to R. Harasawa et al. Microbiol. Immunol. 36, pp 661-665,1992.

DETAILED DESCRIPTION OF THE INVENTION

1. ARGININE DEIMINASE

Accordingly, the present invention includes a novel protein comprisingSEQ ID NO:2 having arginine deiminase enzyme activity and a nucleic acidmolecule encoding the same. Preferably, the ADI is expressed by a novelgene, comprising the nucleic acid sequence of SEQ ID NO:1, that isisolated from M. arthritidis. The present invention also includesmethods of making and using the same. In order for the reader to betterappreciate the description to follow, the following terms are explained.

The nucleotide sequence of SEQ ID NO:1 is presented in the form of adeoxyribonucleic acid or DNA sequence. However, the artisan willunderstand that the nucleotide sequence of SEQ ID NO:1 can also beprepared in the form of an RNA molecule, as necessary. Further, thenucleotides comprising the DNA or RNA molecule can also be in the formof nucleotide derivatives or analogs, such as, for example, those listedat 37 C.F.R. § 1.822(p)(1), the disclosure of which is incorporated byreference herein in its entirety. In addition, the invention alsoencompasses the complement of the nucleotide sequences according to theinvention. The artisan will appreciate the fact that the scope of theinvention also includes alternate codons which can code for the sameamino acid due to the degenerate nature of the genetic code.

"Transfection" refers to the taking up of an expression vector by a hostcell, whether or not any coding sequences are in fact expressed.Numerous methods of transfection are known to the ordinarily skilledartisan. For example, transfection is accomplished in the presence of anexpression vector and high concentrations of CaPO₄, by electroporation,by use of a phage or viral expression vector for insertion into a hostcell, by mechanical insertion of nucleic acid, and even by culturing thehost cells in the presence of unpackaged nucleic acid fragments.Successful transfection is generally recognized when any indication ofthe operation of the vector of interest occurs within the host cell.

"Transformation" describes the introduction of a nucleic acid into anorganism so that the nucleic acid is replicable, either as anextrachromosomal element or by integration in the host chromosome.Depending on the host cell used, transformation is accomplished usingart known methods appropriate to particular host cells. The calciumtreatment employing calcium chloride, as described by Cohen, S. N. Proc.Natl. Acad. Sci. (USA), 69: 2110 (1972) and Mandel et al., J. Mol. Biol.53:154 (1970), is generally used for prokaryotes or other cells that areencapsulated within cellular walls (e.g., many bacterial and/or plantcells). For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham, F. and van der Eb, A.,Virology, 52: 456-457 (1978) is preferred. General aspects of mammaliancell host system transformations have been described in U.S. Pat. No.4,399,216 issued Aug. 16, 1983. Transformations into yeast are typicallycarried out according to the method of Van Solingen, P., et al., J.Bact., 130: 946 (1977) and Hsiao, C. L., et al., Proc. Natl. Acad. Sci.(USA) 76: 3829 (1979). However, any other art-known methods forintroducing nucleic acid, e.g., DNA, into cells, such as, for example,by nuclear injection or by protoplast fusion, may also be used.

As used herein, the term "complementary" with respect to a nucleic acidrefers to the (using Watson-Crick base pairing) opposite strand producedwhen a first nucleic acid molecule is replicated using that molecule asa template, to form a new, second nucleic acid strand that iscomplementary to the first. In one aspect of the invention, two nucleicacid molecules are considered to be complementary, each to the other,when they hybridize or bind together under stringent conditions.

The expression "hybridize under stringent conditions" to describe thehybridization of nucleic acid molecules encompassed within the scope ofthis invention refers to hybridizing under conditions of highhybridization specificity, e.g., low ionic strength and high temperaturefor washing. Such stringent conditions include, for example,hybridization with 0.15M NaCl/0.015M sodium citrate/0.1% NaDodSO₄ at 50°C., or alternatively, in the presence of denaturing agents such asformamide, for example, 50% (vol/vol) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate, at 42° C. forhybridization. "Hybridize under low stringency" refers to hybridizingunder conditions of reduced hybridization specificity. Such conditionsinclude, simply by way of example, hybridizing at 42° C. in 20%formamide, 5×SSC, 50 mM sodium phosphate pH 6.8, 0.1% sodiumpyrophosphate, 5×Denhardt's solution, and 50 μg/ml salmon sperm DNA, andwashing with 2×SSC, 0.1% SDS at 42° C.

Additionally, specific mutations can be introduced into the argininedeiminase gene of the present invention using "site-directedmutagenesis". This is a technique standard in the art, and is conducted,e.g., using a synthetic oligonucleotide primer complementary to asingle-stranded phage DNA to be mutagenized except for limitedmismatching, representing the desired mutation. Such mutations mayinclude, for example, the deletion, insertion, or substitution of thecodons expressing naturally occurring amino acids. Such mutations mayconfer altered protein characteristics, which may, for example, improveand/or alter the oxidative, thermal, and/or pH stability of the protein.Briefly, in this method, the synthetic oligonucleotide is used as aprimer to direct synthesis of a strand complementary to the phage, andthe resulting double-stranded DNA is transformed into a phage-supportinghost bacterium. Cultures of the transformed bacteria are plated on agar,permitting plaque formation from single cells that harbor the phage.Usually from about 50 up to about 90% of the new plaques will containthe phage having, as a single strand, the mutated form. The plaques arehybridized with kinased synthetic primer at a temperature that permitshybridization of an exact match, but at which the mismatches with theoriginal strand are sufficient to prevent hybridization. Plaques thathybridize with the probe are then selected and cultured, and the DNA isrecovered. Thus, the artisan will appreciate that the nucleotidesequence of SEQ ID NO:1 can be conveniently subjected to mutagenesis byart known techniques, e.g., by nucleotide substitution, to produceuseful variant alleles. Simply by way of example, the nucleotidesequence of SEQ ID NO:1 has been variously prepared with substitutionsproviding a T at nucleotide 39, a C at nucleotide 104, an A atnucleotide 206, a G at nucleotide 337, an A at nucleotide 729, a C atnucleotide 830, a C at nucleotide 1023, an A at nucleotide 6, a T atnucleotide 15, a C at nucleotide 18 and/or combinations thereof. Inparticular, the specific substitution at 337 would change the threonineto alanine.

"Operably linked" refers to a juxtaposition of components, e.g., aregulatory region and an open reading frame, such that the normalfunction of the components can be performed. Thus, an open reading framethat is "operably linked" to control sequences refers to a configurationwherein the coding sequence can be expressed under the control of thesesequences and wherein the DNA sequences being linked are contiguous and,in the case of a secretory leader, contiguous and in reading phase. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, then, for example, synthetic oligonucleotideadaptors or linkers are used in accord with conventional practice.

"Control Sequences" refers to nucleic acid sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, and possibly, other as yet poorly understood sequences.Eukaryotic cells are known to utilize, for example, such controlsequences as promoters, polyadenylation signals, and enhancers, to namebut a few.

"Expression system" or "expression vector" refers to nucleic acidsequences containing a desired coding sequence and control sequences inoperable linkage, so that hosts transformed with these sequences arecapable of producing the encoded proteins. To effect transformation, theexpression system may be included on a vector; however, the relevantnucleic acid molecule may then also be integrated into the hostchromosome.

As used herein, "cell," "cell line," and "cell culture" are usedinterchangeably and all such designations include progeny. Thus,"transformants" or "transformed cells" includes the primary subject celland cultures derived therefrom without regard for the number oftransfers. It is also understood that all progeny may not be preciselyidentical in genomic content, due to deliberate or inadvertentmutations. Mutant progeny that have the same functionality as screenedfor in the originally transformed cell are included. Where distinctdesignations are intended, it will be clear from the context.

The vectors disclosed herein are suitable for use in host cells over awide range of prokaryotic and eukaryotic organisms. In general,prokaryotes are preferred for the initial cloning of DNA sequences andconstruction of the vectors useful in the invention. For example, E.coli K12 strain MM 294 (ATCC No. 31,446) is particularly useful. Othermicrobial strains, simply by way of example, that may be used include E.coli strains such as E. coli B and E. coli X1776 (ATCC No. 31,537).Prokaryotes may also be used for expression. The aforementioned strains,as well as, e.g., E. coli strains W3110 (F-, lambda-, prototrophic, ATCCNo. 27,325), K5772 (ATCC No. 53,635), and SR101, bacilli such asBacillus subtilis, and other enterobacteriaceae such as Salmonellatyphimurium or Serratia marcesans, and various pseudomonas species, maybe used.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences that are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (see, e.g., Bolivar et al., 1977, Gene, 2: 95). pBR322 containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. Similarly, the pUC plasmidsprovide convenient cloning vectors with DNA molecules for selection andreplication (Yanisch-Perron, et al., 1985, Gene 33:103-119, thedisclosure of which is incorporated by reference herein in itsentirety). The pBR322 plasmid, or other microbial plasmid or phage, mustalso contain, or be modified to contain, promoters that can be used bythe microbial organism for expression of its own proteins.

"Plasmids" are designated by a lower case "p" preceded and/or followedby capital letters and/or numbers. The starting plasmids herein arecommercially available, are publicly available on an unrestricted basis,or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

Those promoters most commonly used in recombinant DNA constructioninclude the beta -lactamase (penicillinase) and lactose promoter systems(Chang et al., 1978 Nature, 375: 615; Itakura et al., 1977, Science,198: 1056; Goeddel et al., 1979, Nature, 281: 544) and a tryptophan(trp) promoter system (Goeddel et al., 1980, Nucleic Acids Res., 8:4057; EPO Appl. Publ. No. 0036,776). While these are the most commonlyused, other microbial promoters have been discovered and utilized, anddetails concerning their nucleotide sequences have been published,enabling a skilled worker to ligate them functionally with art knownvectors, e.g., plasmid vectors.

Simply by way of example, transcriptional regulation in E. coli may beachieved with any of the following inducible promoters: lac, trp, phoA,araBAD, T7, and derivatives of the lambda P_(L) and P_(R) promoters aswell as others well known to the art (e.g., Makrides, 1996, Microbiol.Rev. 60:512-538, the disclosure of which is incorporated by referenceherein in its entirety). Preferably, the promoter is the O_(L) /P_(R)hybrid promoter, described by Scandella et al., in co-owned U.S. Pat.No. 5,162,216, the disclosure of which is incorporated by referenceherein in its entirety, is employed.

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures,may also be used. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example (Stinchcomb et al., 1979,Nature, 282: 39; Kingsman et al., 1979, Gene, 7: 141; Tschemper et al.,1980, Gene, 10: 157), is commonly used. This plasmid already containsthe trp1 gene that provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, for example, ATCC No.44,076 or PEP4-1 (Jones, 1977, Genetics, 85: 12). The presence of thetrp1 lesion as a characteristic of the yeast host cell genome thenprovides an effective environment for detecting transformation by growthin the absence of tryptophan.

The Pichia pastoris expression system has been shown to achieve highlevel production of several proteins (Cregg, J. M. et al., 1993,Bio/Technology 11: 905-910, the disclosure of which is incorporated byreference herein in its entirety) and may be employed to express ADI asa soluble protein in the cytoplasm of Pichia pastoris.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., J. 1980, Biol. Chem., 255:2073) or other glycolytic enzymes (Hess et al., 1968, J. Adv. EnzymeReg., 7: 149; Holland et al., 1978, Biochemistry, 17: 4900), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3' of the sequencedesired to be expressed to provide polyadenylation of the MRNA andtranscription termination. Other promoters, which have the additionaladvantage of transcription controlled by growth conditions, are thepromoter region for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, andenzymes responsible for maltose and galactose utilization. Any plasmidvector containing yeast-compatible promoter, origin of replication andtermination sequences is suitable.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

Satisfactory amounts of protein are produced by cell cultures; however,refinements, using a secondary coding sequence, serve to enhanceproduction levels even further. One secondary coding sequence comprisesdihydrofolate reductase (DHFR) that is affected by an externallycontrolled parameter, such as methotrexate (MTX), thus permittingcontrol of expression by control of the methotrexate concentration.

Although any suitable strain of M. arthritidis can be employed as asource of the novel gene according to the invention, preferably, the M.arthritidis strain that is employed is the strain deposited in theAmerican Type Culture Collection ("ATCC") as accession number 14152.

A gene capable of expressing the novel arginine deiminase enzymeaccording to the invention is preferably isolated from the M.arthritidis genome by primer directed nucleic acid amplification andthereafter cloned into any suitable screening vector and expressionsystem (Molecular Cloning: A Laboratory Manual, Second Edition, J.Sambrook, E. F. Fritsch and T Maniatis, Eds., Cold Spring Harbor Press,1989). The artisan will appreciate that any appropriate art-knownnucleic acid amplification method, utilizing suitable primers, can beemployed. Preferably, the amplification method is the polymerase chainreaction, as described, simply by way of example, by Mullis, in U.S.Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, the disclosures of whichare incorporated herein by reference in their entireties.

The artisan will also appreciate that any plasmid, phage or cosmidexpression vector, inserted into the respective host cells, may beemployed to express, screen and identify an amplified nucleic acidfragment encoding the arginine deiminase of the invention. Preferably,an Escherichia coil expression system is employed. More preferably aGX6712/pGX9401 Escherichia coli expression system is employed.

The primers used for amplification of the ADI gene according to theexamples provided hereinbelow were oligonucleotides having the sequencesof SEQ ID NOs 3 and 4, respectively. These primers successfullyamplified the entire gene. As indicated in the Examples below, theprimer of SEQ ID NO:3 differed from the corresponding N-terminalencoding region of the isolated gene (SEQ ID NO:1) by three basesubstitutions which did not alter the encoded peptide sequence. Thethree base substitutions, the numbering of which is based on thenumbering of SEQ ID NO:1, are as follows: at base 6 is A is substitutedfor T; at base 15 T is substituted for C; and at base 18 C issubstituted for T.

Thus, the successful amplification of the entire gene having a sequencecomprising SEQ ID NO:1 was surprising, since, as a result of theaforementioned mismatches, relative to the naturally occurring Nterminal sequence of the isolated ADI gene, the SEQ ID NO:3 primerannealed poorly to the target fragment, thus providing an explanationfor the very weak PCR signal that resulted from amplification using theprimer of SEQ ID NO:3.

The artisan will also appreciate that a primer based on the exactnaturally occurring N-terminal sequence of SEQ ID NO:1 will also readilyserve, with even better efficacy, at amplifying the ADI gene accordingto the invention.

Clones provided according to the examples hereinbelow include plasmidpEN232 comprising the ADI gene in expression vector pGX 9401. Theinitial two PCR isolates of the M. arthritidis ADI gene were also clonedinto plasmid pBluescript II SK(-) (Stratagene Cloning Systems, La Jolla,Calif.) and designated pEN241 and pEN242. The N-terminal fragmentcloning of an ADI gene segment into pBluescript II SK(-), described inExample 1B, included two independently analyzed clones designated pEN245and pEN246. Independent transformations of E. coli DH5-alpha (GIBCO BRL,Gaithersburg, Md.) with plasmids pEN241, pEN242, pEN245 and pEN246produced E. coli clones designated EN243, EN244, EN247 and EN248,respectively.

Once a transfected or transformed host cell is obtained, a nucleic acidmolecule that includes a sequence according to SEQ ID NO:1 is readilyproduced by culturing the host cell, and extracting and isolating thenucleic acid as desired, by methods well known to the art. Depending onthe degree of purity desired, the extracted nucleic acid may beisolated, or where desired, substantially isolated by art known methods,to be free or substantially free of contaminating host cell proteins andnucleic acids. Similarly, host cells expressing the arginine deiminaseprotein encoded by the expression vectors according to the invention arecultured by methods suitable for the selected host cell.

For example, host cells are cultured until desired cell densities areachieved, and then the cells are separated from the growth medium andthe protein is extracted and thereafter renatured according to art-knownmethods. In particular, the cells are separated from the culture mediumto form a cell paste. The cell paste is then re-suspended and thendisrupted by standard methods, e.g., mechanical, ultrasonic and/orchemical disruption. Preferably, the cells are disrupted by processingin a Microfluidizer (Microfluidics Corp. Newton Mass.) followed bywashing with a suitable surfactant, such as, for example, Triton X-100.

The resulting homogenate is denatured with guanidine HCl, 6M and thendiluted into refolding buffer (e.g., 10 mM K₂ PO₄, pH 7.0), particulatesremoved, e.g., by centrifugation, followed by purification of thesupernatant by standard methods, e.g., by Q Sepharose columnchromatography, to provide substantially purified arginine deiminase,e.g., with a purity of about 60% and having a specific activity rangingfrom about 3 to about 25 IU/mg, or more, and preferably from about 5 toabout 20 IU/mg. Additional concentration of the ADI can be achievedusing a Centriprep-10, Amicon, Inc. Beverly, Mass.. Other similarlyoperating columns can also be used if desired.

2. NON-ANTIGENIC POLYMERS

In order to form the polymer-arginine deiminase conjugates of thepresent invention, polymers such as poly(alkylene oxides) (PAO's) areconverted into activated forms, as such term is known to those ofordinary skill in the art. Thus, one or both of the terminal polymerhydroxyl end-groups, (i.e. the alpha and omega terminal hydroxyl groups)are converted into reactive functional groups which allows covalentconjugation. This process is frequently referred to as "activation" andthe product is called an "activated poly(alkylene oxide)". Polymerscontaining both alpha and omega linking groups are referred to asbis-activated polyalkylene oxides. Other substantially non-antigenicpolymers are similarly "activated" or functionalized. Among thesubstantially non-antigenic polymers, mono-activated polyalkylene oxides(PAO's), such as monomethoxy-polyethylene glycols are preferred. Inalternative embodiments, homobifunctional bis-activated polymers such asbis-succinimidyl carbonate activated PEG are preferred.

The activated polymers are thus suitable for reacting with argininedeiminase and forming ADI-polymer conjugates wherein attachmentpreferably occurs at either the amino terminal α-amino group or ε-aminogroups of lysines found on the ADI.

In one preferred aspect of the invention, carbamate (urethane) linkagesare formed using the ADI ε amino groups and the activated polyalkyleneoxides. Preferably, the carbamate linkage is formed as described incommonly owned U.S. Pat. No. 5,122,614, the disclosure of which ishereby incorporated by reference. This patent discloses the formation ofmono- and bis- N-succinimidyl carbonate derivatives of polyalkyleneoxides (SC-PEG). Alternatives include para-nitrophenyl carbonate andcarbonyl imidazole activated polymers.

In another aspect of the invention, polymers activated withamide-forming linkers such as cyclic imide thione-activated polyalkyleneoxides, succinimidyl esters or the like are used to effect the linkagebetween the arginine deiminase and polymer terminal groups, see forexample, U.S. Pat. No. 5,349,001 to Greenwald, et al., the disclosure ofwhich is incorporated herein by reference. Still other aspects of theinvention include using other activated polymers to form covalentlinkages of the polymer with the arginine deiminase via ε amino or othergroups. For example, isocyanate or isothiocyanate forms of terminallyactivated polymers can be used to form urea or thiourea-based linkageswith the lysine amino groups. PEG-dialdehyde can also be reacted withthe arginine deiminase followed by reduction with NaCNBH₃ to form asecondary amine linkage.

Suitable polymers will vary substantially by weight, however polymershaving molecular weights ranging from about 200 to about 60,000 areusually selected for the purposes of the present invention. Molecularweights of from about 1,000 to about 40,000 are preferred and 2,000 toabout 20,000 are particularly preferred.

The polymeric substances included are also preferably water-soluble atroom temperature. A non-limiting list of such polymers includepolyalkylene oxide homopolymers such as polyethylene glycol (PEG) orpolypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof, provided that the water solubility of theblock copolymers is maintained.

As an alternative to PAO-based polymers, effectively non-antigenicmaterials such as dextran, polyvinyl pyrrolidones, polyacrylamides,polyvinyl alcohols, carbohydrate-based polymers and the like can beused. Indeed, the activation of alpha and omega terminal groups of thesepolymeric substances can be effected in fashions similar to that used toconvert polyalkylene oxides and thus will be apparent to those ofordinary skill. Those of ordinary skill in the art will realize that theforegoing list is merely illustrative and that all polymer materialshaving the qualities described herein are contemplated. For purposes ofthe present invention, "effectively non-antigenic" means all materialsunderstood in the art as being nontoxic and not eliciting an appreciableimmunogenic response in mammals.

3. REACTION CONDITIONS

Conjugation reactions, sometimes referred to as pegylation reactions,are generally carried out in solution with from about an equimolar toabout a several fold molar excess of activated polymer. Preferably, themolar excess of activated polymer is about 50-fold, or greater. One wayto maintain the arginine deiminase bioactivity is to substantially avoidincluding those arginine deiminase lysines associated with the activesite in the polymer coupling process. Given the usually non-specificnature of the coupling reaction, this theoretical step is oftendifficult to achieve in practice. The process of the present invention,however, provides arginine deiminase conjugates having high levels ofretained activity by using arginine deiminase obtained from M.arthritidis, which has a substantial increase in the number of availableof lysines for polymer attachment, and avoiding the use of anexcessively high molar excess, e.g., more than about 100 fold, ofactivated polymer during the conjugation reactions.

Thus, the conjugation conditions include reacting arginine deiminaseobtained from M. arthritidis with a suitably activated substantiallynon-antigenic polymer such as SC-PEG in a suitable buffer solution in aratio of activated polymer to arginine deiminase of from about 50 toabout 100 fold.

The conjugation reaction is carried out under relatively mild conditionsto avoid inactivating the arginine deiminase. Mild conditions includemaintaining the pH of the reaction solution in the range of 6-8 and thereaction temperatures within the range of from about 0-30° C. andpreferably at about 4° C. for about one hour. Suitable buffers includebuffer solutions able to maintain the preferred pH range of 6-8 withoutinterfering with the conjugation reaction. A non-limiting list ofsuitable buffers includes, e.g., phosphate buffer, citrate buffer,acetate buffer.

Although the reaction conditions described herein may result in someunmodified arginine deiminase, the unmodified arginine deiminase can bereadily recovered and recycled into future batches for additionalconjugation reactions.

The conjugation reactions of the present invention initially provide areaction mixture or pool containing arginine deiminase conjugates havingfrom about 16 to about 22 strands of polymer per subunit of enzyme (31total lysines), unreacted arginine deiminase, if any, and unreactedpolymer. After the unreacted species have been removed, compositionscontaining the arginine deiminase-polymer conjugates are recovered.These compositions have at least about 20% of the biological activityassociated with the native or starting arginine deiminase as measuredusing an assay such as that described below in Example 3. In preferredaspects of the invention, however, the conjugates have at least about30% of the biological activity associated with starting argininedeiminase and most preferably, the conjugates have at least about 90% ofthe biological activity associated with starting arginine deiminase.

A representative conjugation reaction is set forth below:

An about 50 fold molar excess of activated polymer is dissolved in WaterFor Injection (pH approximately 6.0) and then added to an M. arthritidisarginine deiminase solution adjusted to about pH 8.0 with a suitablebuffer such as a phosphate or borate buffer. The reaction is allowed toincubate at about 4° C., at about pH 8.0, for a suitable time, such asabout 1 hour, with continuous gentle mixing. Thereafter, the conjugationreaction is stopped, for example with a several-fold molar excess ofarginine or glycine. The unmodified arginine deiminase present in thereaction pool, if any, after the conjugation reaction has been quenched,can be recovered for recycling into future reactions using ion exchangeor size exclusion chromatography or similar separation techniques.Preferably, solutions containing the conjugates of the present inventioncontain less than about 5% unmodified arginine deiminase.

If desired, the arginine deiminase-polymer conjugates are isolated fromthe reaction mixture to remove high molecular weight species, andunmodified arginine deiminase. The separation process is commenced byplacing the mixed species in a buffer solution containing from about1-10 mg/ml of the arginine deiminase-polymer conjugates. Suitablesolutions have a pH of from about 6.0 to about 9.0 and preferably fromabout 7.5 to about 8.5. The solutions preferably contain one or morebuffer salts selected from KCl, NaCl, K₂ HPO₄, KH₂ PO₄, Na2HPO₄, NaH₂PO₄, NaHCO₃, NaBO₄, and NaOH. Sodium phosphate buffers are preferred.

Depending upon the reaction buffer, the arginine deiminase polymerconjugate solution may first have to undergo bufferexchange/ultrafiltration to remove any unreacted polymer. For example,the PAO-Arginine deiminase conjugate solution can be ultra-filteredacross a low molecular weight cut-off (10,000 to 30,000 Dalton) membraneto remove most unwanted materials such as unreacted polymer,surfactants, if present, or the like.

Fractionation of the ADI-polymer conjugates, if desired, can also becarried out using an anion exchange chromatography medium. Such mediaare capable of selectively binding PAO-arginine deiminase conjugates viadifferences in charge which vary in a somewhat predictable fashion. Forexample, the surface charges of ADI is determined by the number ofavailable charged amino acids on the surface of the protein. Of thesecharged amino acids, lysine residues serve as the point of potentialattachment of polyalkylene oxide conjugates. Therefore, argininedeiminase conjugates will have a different charge from the other speciesto allow selective isolation. The use of strongly polar anion exchangeresins such as quaternary amine anion exchange resins are especiallypreferred for the method of the present invention. Included among thecommercially available quaternary anion exchange resins suitable for usewith the present invention are Q-HD, QA TRISACRYL® and QMA-SPHEROSIL®,quaternary amine resins coated onto a polymer matrix, manufactured byIBF of Garenne, France, for Sepracor of Marlborough, Mass.; TMAE650M®, atetramethylamino ethyl resin coated onto a polymer matrix, manufacturedby EM-Separators of Gibbstown, N.J.; QAE550C®, and SUPERQC®, each aquaternary amine resin coated onto a polymer matrix and manufactured byTosoHaas of Montgomeryville, Pa. QMA Accell, manufactured by Milliporeof Milford, Mass. and PEI resins manufactured by JT Baker ofPhillipsburg, N.J., may also be used. Other suitable anion exchangeresins e.g. DEAE resins can also be used.

For example, the anion exchange resin is preferably packed in a columnand equilibrated by conventional means. A buffer having the same pH andosmolality as the polymer conjugated arginine deiminase solution isused. The elution buffer preferably contains one or more salts selectedfrom KCl, NaCl, K₂ HPO₄, KH₂ PO₄, Na₂ HPO₄, NaH₂ PO₄, NaHCO₃, NaBO₄ and(NH₄)₂ CO₃. The conjugate-containing solution is then adsorbed onto thecolumn with the high molecular weight species and unreacted polymer notbeing retained. At the completion of the loading, a gradient flow of anelution buffer with increasing salt concentrations is applied to thecolumn to elute the desired fraction of polyalkylene oxide-conjugatedarginine deiminase. The eluted pooled fractions are preferably limitedto uniform mono- and bis-arginine deiminase polymer conjugates after theanion exchange separation step. Any unconjugated arginine deiminasespecies can then be back washed from the column by conventionaltechniques. If desired, the arginine deiminase species can also beseparated via additional ion exchange chromatography or size exclusionchromatography. The temperature range for elution is between about 4° C.and about 25° C. Preferably, elution is carried out at a temperature offrom about 6° C. to about 22° C. Fraction collection may be achievedthrough simple time elution profiles.

4. METHODS OF TREATMENT

Another aspect of the present invention provides methods of treatmentfor various medical conditions in mammals. The methods includeadministering an effective amount of arginine deiminase-polymerconjugates which have been prepared as described herein to a mammal inneed of such treatment. The conjugates are useful for, among otherthings, treating arginine deiminase-susceptible conditions or conditionswhich would respond positively or favorably as these terms are known inthe medical arts to arginine deiminase-based therapy. Thus, withoutlimitation, the arginine deiminase conjugates can be used to treatconditions, including, carcinomas deficient in the enzymeargininosuccinate synthetase, e.g., melanoma (Sugimura et al., 1992,Melanoma Res. 2:191-196) and nitric oxide related conditions, e.g.,conditions that may be treated or ameliorated by modulation of nitricoxide synthase (Nagasaki et al, 1996, J. Biol. Chem. 271:2658-2662; Xiaet al., 1996, Proc. Natl. Acad. Sci. USA 93:6770-6774) and certaindietary applications, e.g., modulating the therapeutic effects of lowprotein diets (Narita et al., 1995, Proc. Natl. Acad. Sci. USA92:4552-4556).

The amount of the arginine deiminase-polymer conjugate administered totreat the conditions described above is based on the arginine deiminaseactivity of the polymeric conjugate. It is an amount that is sufficientto significantly effect a positive clinical response. The maximal dosefor mammals including humans is the highest dose that does not causeclinically-important side effects. For purposes of the presentinvention, such clinically important side effects are those which wouldrequire cessation of therapy such as, for example, hypersensitivityreactions and/or other immunogenic reactions.

Naturally, the dosages of the arginine deiminase-based compositions willvary somewhat depending upon the arginine deiminase moiety and polymerselected. In general, however, the conjugate is administered in amountsranging from about 300 to about 3000 IU/m² of arginine deiminase perday, based on the mammal's condition. The range set forth above isillustrative and those skilled in the art will determine the optimaldosing of the conjugate selected based on clinical experience and thetreatment indication.

The ADI-polymer conjugates of the present invention can be included inone or more suitable pharmaceutical compositions for administration tomammals. The pharmaceutical compositions may be in the form of asolution, suspension, tablet, capsule or the like, prepared according tomethods well known in the art. It is also contemplated thatadministration of such compositions will be chiefly by the parenteralroute although oral or inhalation routes may also be used depending uponthe needs of the artisan.

EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention.

Example 1

Expression of M. arthritidis ADI Gene in E. Coli

A. Isolation and Cloning of ADI Gene

M. arthritidis strain 14152 was obtained from the American Type CultureCollection. The arginine deiminase gene of M. arthritidis was amplifiedby a polymerase chain reaction (PCR) using the primer pair5'GGCAATCGATGT CTGTATTTGACAGTA-3'(SEQ ID NO:3) and5'-TGAGGATCCTTACTACCACTTAACATCTTTACG-3'(SEQ ID NO:4) derived from thepublished sequence of M. arginini LBIF (Ohno, T. et al. 1990) "Cloningand Nucleotide Sequence of the Gene Encoding Arginine Deiminase ofMycoplasma arginini" Infect Immun., 58: 3788-3795, the contents of whichare incorporated herein by reference.

The PCR amplification was conducted by standard methods (as reviewed bySaiki et al., 1989, PCR Technology, pages 7-16; Ed. Henry A. Erlich,Stockton Press) with the following parameters.

The reaction was conducted in a volume of 100 microliters, with a PCRbuffer of 10 millimolar Tris-HCl, pH 8.3, 50 millimolar KCl, 2millimolar MgCl₂, and 200 μM of each deoxynucleotide triphosphate (dATP,dCTP, dGTP and dTTP) and 2.5 units of Taq DNA polymerase (from PerkinElmer). Thirty cycles of amplification were carried out in a PerkinElmer PCR system 9600 thermal cycler, set to denature at 94 degrees C.for 60 seconds, anneal at 37° C. for 180 seconds and extend at 72degrees C. for 120 seconds.

After PCR amplification, two rather faint bands representing amplifiedfragments were observed on agarose gel analysis at 1.4 kb and 1.2 kb.Samples of each fragment, respectively, were excised from the gel,purified and cloned as a ClaI-BamHI fragment directly into expressionplasmid pGX9401 in the manner disclosed by Filpula et al. in"Engineering of Immunoglobulin Fc and Single Chain Fv Proteins inEscherichia coli" in Antibody Expression and Engineering (H. Y. Wang andT. Imanaka, eds.) American Chemical Society, pp 70-85 the contents ofwhich are incorporated herein by reference. Both fragments weresequenced, and the 1.2 kb fragment was confirmed as encoding ADI by itspartial homology to previously known genes encoding enzymes with ADIenzyme activity.

The five TGA codons in the isolated ADI gene which encode tryptophan inMycoplasma were changed to TGG codons by oligonucleotide-directedmutagenesis according to the method of Sayers et al. Biotechniques 13:592-596 (1992), the disclosure of which is incorporated herein byreference, prior to gene expression in E. coli. The GX6712/pGX9401 E.coli expression system used was the same as that described in theaforementioned Filpula et al. reference. Recombinant ADI was expressedin inclusion bodies at levels of 10% of the total cell protein.

B. Confirmation of N-Terminal Sequence of ADI Gene

In order to confirm the N-terminal region of the M. arthritidis genecorresponding to PCR primer SEQ ID NO:3, an independent PCRamplification of the N-terminal region was conducted using theestablished DNA sequence data from the first PCR. The technique employedwas that of "inverse PCR" as described by H. Ochman et al., 1990, (PCRPROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, Inc.,Eds., M. A. Innis, D. H. Gelfand et al.).

The inverse PCR was conducted using with PCR primers5'-CTAAAACGGTTTCTAGTTCACC-3' (SEQ ID NO:5) and 5'- AGCGTGGAATTAATGTTGTTG-3' (SEQ ID NO:6). Two micrograms of genomic M. arthritidis DNA wasdigested with restriction endonuclease Sau 3A, then fragments werecircularized by treatment with T4 DNA ligase. This DNA preparation wasthen subjected to PCR amplification using SEQ ID NOs 5 and 6. Theamplified DNA was cloned and analyzed by DNA sequencing. DNA sequenceanalysis confirmed the assignment of serine as the amino acid followingthe initiation methionine (which is predicted to be post-translationallyremoved). Three silent base changes were also noted: Base 6 is A ratherthan T; Base 15 is T rather than C; Base 18 is C rather than T. Inaddition, Base 39 is T rather than C in this cloned sequence. None ofthese changes alters the translated protein sequence.

The three base differences between the N-terminal coding region of theconfirmed genomic sequence and the SEQ ID NO:3 primer employed toisolate the gene are believed to account for the rather faint bands, asdiscussed above, that were produced when the PCR product was analyzed bygel electrophoresis. The three base differences are believed to haveresulted in a poor annealing between the primer and the N-terminalcoding region, resulting in a weak PCR signal observed on gelelectrophoresis. Thus, given this difference and the weak PCR signal,the successful amplification of the entire ADI gene by PCR requiredcareful selection of PCR conditions and therefore successful isolationof the gene represented by SEQ ID NO:1 was unexpected.

Example 2

Renaturation and Purification of Recombinant ADI

In this example, the ADI protein obtained as result of Example 1 isrenatured according to the techniques reported by Misawa et al. , withminor modifications (Misawa et al., 1994 "High-Level Expression ofMycoplasma arginine deiminase in Escherichia coli and Its EfficientRenaturation As An Antitumor Enzyme" in J. Biotechnol. 36: 145-155, thecontents of which are incorporated herein by reference in its entirety).

To begin, 100 grams of cell paste is resuspended in 800 ml of 10 mM K₂PO₄, pH 7.0, 1 mM EDTA (buffer 1) and the cells are disrupted by twopasses in a Microfluidizer (Microfluidics Corp. Newton Mass.). TritonX-100 is added to achieve a final concentration of 4% (v/v). Thehomogenate is stirred for 30 minutes at 4 degrees C. and is thencentrifuged for 30 minutes at 13,000 g. The pellet is collected andre-suspended in one liter of buffer 1 containing 0.5% Triton X-100. Thesolution is diafiltered against 5 volumes of denaturation buffer (50 mMTris HCl, pH 8.5, 10 mM DTT) using hollow fiber cartridges with 100 kDretention rating (Microgon Inc. Laguna Hills, Calif.). Guanidine HCl isadded to achieve a final concentration of 6M and the solution is stirredfor 15 minutes at 4 degrees C. The solution is then diluted 100-foldinto refolding buffer (10 mM K O₄, pH 7.0) and stirred for 48 hours at15 degrees C. Particulates are removed by centrifugation at 13,000 g.The resultant supernatant is concentrated on a Q Sepharose Fast Flow(Pharmacia, Inc. Piscataway, N.J.) column pre-equilibrated in refoldingbuffer. ADI is eluted using refolding buffer containing 0.2M NaCl. Thepurification procedure yields ADI protein which is >95% pure asestimated SDS-PAGE analysis. About eight grams of pure renatured ADIprotein are produced from 1 kilogram of cell paste, which corresponds toa yield of 200 milligrams of ADI per liter of fermentation.

Example 3

Arginine Deiminase Assay

ADI activity was determined by a minor modification of the methoddescribed by Oginsky et al. in "Isolation and Determination of Arginineand Citrulline" Methods Enzymology 3: 639-643 (1957), the disclosure ofwhich is incorporated herein by reference. Ten microliter samples in0.1M Na₂ PO₄, pH 7.0 (BUN assay buffer) were placed in a 96 wellmicrotiter plate, 40 microliters of 0.5M arginine in BUN assay bufferwas added and the plate was covered and incubated at 37° C. for 15minutes. 200 microliters of complete BUN reagent (Sigma Diagnostics) wasadded and the covered plate was incubated for 10 minutes at 100° C. Theplate was cooled to 22° C. and analyzed at 490 nm by a microtiter platereader (Molecular Devices, Inc.). One IU is the amount of enzyme whichconverts 1 micromole of L-arginine to L-citrulline per minute. Proteinconcentrations were determined using Pierce Coomassie Plus Protein AssayReagent (Pierce Co., Rockford, Ill.) with bovine serum albumin asstandard. The specific enzyme activity of the purified ADI preparationswas determined to be about 3 to about 30 IU/mg.

Example 4

In this example, succinimidyl carbonate-activatedmonomethoxypolyethylene glycol, molecular weight 12,000, was used tomodify the arginine deiminase obtained as a result of Example 2. Thesuccinimidyl carbonate activated mPEG was prepared in accordance withthe method of the aforementioned U.S. Pat. No. 5,122,614.

A solution of arginine deiminase (0.910 mg in a volume of 1 ml) wasadjusted to pH 8.0 with 100 mM borate buffer. A 50-fold molar excess ofthe activated PEG was dissolved in Water For Injection and then added tothe arginine deiminase. The reaction was incubated at 4° C. for about 1hour with continuous gentle mixing. After 1 hour, the reaction wasstopped with an excess of arginine. The PEG-ADI conjugates werediafiltered through a Centriprep 30 with 15 volumes of 0.1 molar sodiumphosphate, pH 7.0, which was monitored at 220 nm for the presence of thepolymer until<0.05. The reaction products were analyzed as described inExample 3, supra, and found to have about 40% retained ADI activity.

Example 5

The process of Example 4 is repeated except that molecular weight 12,000mPEG activated with an N-acyl thiazolidine is used.

Example 6

The process of Example 4 is repeated except that succinimidylcarbonate-activated monomethoxy-polyethylene glycol, molecular weight5,000, is used.

Example 7

PEG₁₂,000 -ADI INHIBITION OF SK-MEL-2 CELL GROWTH

In this example, the PEG-ADI prepared in accordance with Example 4 wascompared to PEG ADI conjugates made using ADI obtained from M. argininiin a cell growth inhibition assay. The M. arginini ADI was obtained in asimilar fashion as that used to obtain the M. arthritidis ADI exceptthat the M. arginini ADI was purified to a higher extent than the M.arthritidis ADI. In particular, the M. arthritidis ADI was extracted andrefolded to about 60% purity but not processed through anion exchangechromatography. Processing through the anion exchange chromatography isexpected to yield greater than 90% purity of the M. arthritidis ADIenzyme. The PEG-conjugation technique used to make the M.arginini-derived ADI conjugates was the same as that used in Example 4.In both cases, PEG MW 12,000 was used to make the conjugates.

Melanoma cells were growth in Minimum Essential Medium (Eagle) with 0.1mM non-essential amino acids, 1 mM sodium pyruvate and Earle's salts;fetal bovine serum 10%. Following trypsinization, viable cells werecounted by trypan blue exclusion. Cells (10⁴) were added to each well ina total of 100 microliters in 96 well micro-titer plates. PEG₁₂,000 -ADI conjugates were diluted by 2-fold serial dilution in complete media(0.1 ml) and added to each well. Plates were incubated at 37° C. in 5%CO₂ incubator. Cell growth at day 3 was measured by adding 1/10th volumeof "alamar Blue" dye (Alamar Biosciences, Inc. Sacramento, Calif.).After five hours of incubation, the plates were read with a MolecularDevice plate reader at 570-630 nm.

The M. arginini derived PEG-ADI conjugates were found to have an IC₅₀ of0.00015 IU/ml while the M. arthritidis- derived PEG-ADI had an IC₅₀ of0.00010 IU/ml. Thus, when normalized in units of IU/ml, the PEG-ADIderived from M. arthritidis is seen to have 150% of the potency of thePEG-ADI derived from M. arginini.

Example 8

SDS PAGE Analysis

An SDS-PAGE analysis was carried out to compare PEG₁₂,000 -ADIconjugates prepared either with M. arginini-derived ADI or, inaccordance with the present invention, with M. arthritidis-derived ADI.The results indicate that the M. arthritidis derived conjugates had amore uniform distribution of size and had a higher average molecularweight (and therefore had more PEG molecules attached per ADI subunit).While Applicants are not bound by theory, it is believed that the M.arthritidis derived ADI included a greater number of lysines upon whichthe PEG could covalently attach, and, perhaps more importantly, thereare a sufficient number of lysines on this specific ADI which are notassociated with the active site.

Example 9

Sequence Comparisons

In this example, the amino acid sequences and sequence alignment ofvarious arginine deiminases were investigated. Turning to FIG. 3, it isnoted that line "a" represents the M. arthritidis ATCC 14152 ADI used inaccordance with the present invention. Line "b" represents M. argininiLBIF ADI. Line "c" represents M. hominis PG21 ADI and line "d"represents M. orale FERMBP-1970 ADI. The dashes indicate amino acidsidentical to those found in line "a". Dots indicate gaps. The percent ofamino acid sequence identity to M. arthritidis ADI is:

M. arginini--87%

M. hominis--81%

M. orale--83%.

This level of non-homology between the encoded amino acid sequences ofthe respective genes indicates significant differences between theencoded proteins of the respective Mycoplasma species. The sites oflysine substitutions are dispersed and extensive, indicating greatdiversity in potential polymer conjugation sites.

While there have been described what are presently believed to be thepreferred embodiments of the invention, those skilled in the art willrealize that changes and modifications may be made thereto withoutdeparting from the spirit of the invention. It is intended to claim allsuch changes and modifications that fall within the true scope of theinvention. Numerous references are cited in the specification, thedisclosures of which are incorporated by reference in their entireties.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 9    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1230 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: Not Relev - #ant    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (vi) ORIGINAL SOURCE:    #arthritidis) ORGANISM: Mycoplasma    #EN231    (B) INDIVIDUAL ISOLATE:              (C) CELL TYPE: unicellu - #lar organism    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - ATG TCT GTA TTT GAC AGT AAA TTT AAG GGA AT - #T CAT GTC TAT TCA GAA      48    Met Ser Val Phe Asp Ser Lys Phe Lys Gly Il - #e His Val Tyr Ser Glu    #                 15    - ATT GGT GAA CTA GAA ACC GTT TTA GTT CAC GA - #A CCT GGT AAA GAA ATT      96    Ile Gly Glu Leu Glu Thr Val Leu Val His Gl - #u Pro Gly Lys Glu Ile    #             30    - GAT TAC ATT ACC CCA GCT CGT TTG GAT GAA TT - #A TTA TTC TCA GCT ATT     144    Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Le - #u Leu Phe Ser Ala Ile    #        450    - CTA GAA AGC CAC GAT GCA AGA AAA GAA CAC AA - #A GAA TTC GTA GCA GAA     192    Leu Glu Ser His Asp Ala Arg Lys Glu His Ly - #s Glu Phe Val Ala Glu    #    605    - CTT AAA AAG CGT GGA ATT AAT GTT GTT GAA TT - #A GTA GAT CTA ATC GTA     240    Leu Lys Lys Arg Gly Ile Asn Val Val Glu Le - #u Val Asp Leu Ile Val    # 80    - GAA ACC TAT GAT TTA GCA TCA AAA GAA GCT AA - #A GAA AAA CTT TTA GAA     288    Glu Thr Tyr Asp Leu Ala Ser Lys Glu Ala Ly - #s Glu Lys Leu Leu Glu    #                 95    - GAA TTC CTA GAT GAT TCA GTA CCA GTT CTA TC - #A GAC GAA CAC CGT GCT     336    Glu Phe Leu Asp Asp Ser Val Pro Val Leu Se - #r Asp Glu His Arg Ala    #           110    - ACT GTT AAG AAA TTC TTA CAA AGT CAA AAA TC - #A ACA AGA TCA TTA GTT     384    Thr Val Lys Lys Phe Leu Gln Ser Gln Lys Se - #r Thr Arg Ser Leu Val    #       125    - GAA TAC ATG ATC GCA GGG ATC ACT AAA CAC GA - #T TTA AAA ATC GAA TCA     432    Glu Tyr Met Ile Ala Gly Ile Thr Lys His As - #p Leu Lys Ile Glu Ser    #   140    - GAT TTA GAA TTA ATC GTT GAC CCA ATG CCT AA - #C TTG TAC TTC ACT CGT     480    Asp Leu Glu Leu Ile Val Asp Pro Met Pro As - #n Leu Tyr Phe Thr Arg    145                 1 - #50                 1 - #55                 1 -    #60    - GAC CCA TTT GCA TCA GTA GGT AAT GGA GTT AC - #C ATC CAC TAC ATG CGT     528    Asp Pro Phe Ala Ser Val Gly Asn Gly Val Th - #r Ile His Tyr Met Arg    #               175    - TAC AAA GTA AGA CAA CGT GAA ACA TTA TTT AG - #C CGA TTT GTA TTT TCA     576    Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Se - #r Arg Phe Val Phe Ser    #           190    - AAT CAC CCT AAA CTA GTT AAT ACC CCA TGG TA - #C TAC GAC CCT GCT GAA     624    Asn His Pro Lys Leu Val Asn Thr Pro Trp Ty - #r Tyr Asp Pro Ala Glu    #       205    - GGA TTA ACA ATC GAA GGT GGA GAC GTA TTT AT - #C TAC AAT AAC GAT ACT     672    Gly Leu Thr Ile Glu Gly Gly Asp Val Phe Il - #e Tyr Asn Asn Asp Thr    #   220    - TTA GTA GTT GGT GTT TCA GAA AGA ACT GAC TT - #A CAA ACT ATT ACT TTA     720    Leu Val Val Gly Val Ser Glu Arg Thr Asp Le - #u Gln Thr Ile Thr Leu    225                 2 - #30                 2 - #35                 2 -    #40    - TTA GCT AAG AAC ATT AAA GCA AAT AAA GAA TG - #T GAA TTC AAA CGT ATT     768    Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cy - #s Glu Phe Lys Arg Ile    #               255    - GTA GCA ATT AAT GTT CCT AAA TGG ACA AAC CT - #A ATG CAC TTA GAC ACA     816    Val Ala Ile Asn Val Pro Lys Trp Thr Asn Le - #u Met His Leu Asp Thr    #           270    - TGG TTA ACA ATG CTA GAC AAA GAT AAA TTC TT - #A TAC TCA CCT ATT GCA     864    Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Le - #u Tyr Ser Pro Ile Ala    #       285    - AAT GAT GTG TTT AAA TTC TGG GAC TAC GAT TT - #A GTT AAT GGC GGA GAC     912    Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Le - #u Val Asn Gly Gly Asp    #   300    - GCT CCT CAA CCA GTT GAC AAT GGA TTA CCT CT - #A GAA GAC TTA TTG AAA     960    Ala Pro Gln Pro Val Asp Asn Gly Leu Pro Le - #u Glu Asp Leu Leu Lys    305                 3 - #10                 3 - #15                 3 -    #20    - TCA ATC ATT GGT AAG AAA CCT ACT CTA ATT CC - #T ATT GCT GGT GCT GGT    1008    Ser Ile Ile Gly Lys Lys Pro Thr Leu Ile Pr - #o Ile Ala Gly Ala Gly    #               335    - GCT TCA CAA ATC GAT ATT GAA CGT GAA ACC CA - #C TTT GAC GGA ACA AAC    1056    Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr Hi - #s Phe Asp Gly Thr Asn    #           350    - TAC CTA GCT GTA GCT CCT GGA ATT GTT ATT GG - #T TAT GCA CGT AAC GAA    1104    Tyr Leu Ala Val Ala Pro Gly Ile Val Ile Gl - #y Tyr Ala Arg Asn Glu    #       365    - AAA ACA AAT GCC GCT TTA GAA GCT GCA GGA AT - #T ACT GTT CTA CCA TTC    1152    Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Il - #e Thr Val Leu Pro Phe    #   380    - AGA GGA AAC CAA CTT TCA CTT GGA ATG GGA AA - #T GCT CGT TGC ATG TCA    1200    Arg Gly Asn Gln Leu Ser Leu Gly Met Gly As - #n Ala Arg Cys Met Ser    385                 3 - #90                 3 - #95                 4 -    #00    #         1230     GT AAA GAT GTT AAG TGG    Met Pro Leu Ser Arg Lys Asp Val Lys Trp    #               410    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #ACID RESIDUESLENGTH: 410 AMINO              (B) TYPE: AMINO ACID              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: PROTEIN    -     (vi) ORIGINAL SOURCE:    #Arthritidis) ORGANISM: Mycoplasma    #EN231    (B) INDIVIDUAL ISOLATE:              (C) CELL TYPE: unicellu - #lar organism    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Met Ser Val Phe Asp Ser Lys Phe Lys Gly Il - #e His Val Tyr Ser Glu    #                 15    - Ile Gly Glu Leu Glu Thr Val Leu Val His Gl - #u Pro Gly Lys Glu Ile    #            30    - Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Le - #u Leu Phe Ser Ala Ile    #        450    - Leu Glu Ser His Asp Ala Arg Lys Glu His Ly - #s Glu Phe Val Ala Glu    #    605    - Leu Lys Lys Arg Gly Ile Asn Val Val Glu Le - #u Val Asp Leu Ile Val    # 80    - Glu Thr Tyr Asp Leu Ala Ser Lys Glu Ala Ly - #s Glu Lys Leu Leu Glu    #                 95    - Glu Phe Leu Asp Asp Ser Val Pro Val Leu Se - #r Asp Glu His Arg Ala    #           110    - Thr Val Lys Lys Phe Leu Gln Ser Gln Lys Se - #r Thr Arg Ser Leu Val    #       125    - Glu Tyr Met Ile Ala Gly Ile Thr Lys His As - #p Leu Lys Ile Glu Ser    #   140    - Asp Leu Glu Leu Ile Val Asp Pro Met Pro As - #n Leu Tyr Phe Thr Arg    145                 1 - #50                 1 - #55                 1 -    #60    - Asp Pro Phe Ala Ser Val Gly Asn Gly Val Th - #r Ile His Tyr Met Arg    #               175    - Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Se - #r Arg Phe Val Phe Ser    #           190    - Asn His Pro Lys Leu Val Asn Thr Pro Trp Ty - #r Tyr Asp Pro Ala Glu    #       205    - Gly Leu Thr Ile Glu Gly Gly Asp Val Phe Il - #e Tyr Asn Asn Asp Thr    #   220    - Leu Val Val Gly Val Ser Glu Arg Thr Asp Le - #u Gln Thr Ile Thr Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cy - #s Glu Phe Lys Arg Ile    #               255    - Val Ala Ile Asn Val Pro Lys Trp Thr Asn Le - #u Met His Leu Asp Thr    #           270    - Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Le - #u Tyr Ser Pro Ile Ala    #       285    - Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Le - #u Val Asn Gly Gly Asp    #   300    - Ala Pro Gln Pro Val Asp Asn Gly Leu Pro Le - #u Glu Asp Leu Leu Lys    305                 3 - #10                 3 - #15                 3 -    #20    - Ser Ile Ile Gly Lys Lys Pro Thr Leu Ile Pr - #o Ile Ala Gly Ala Gly    #               335    - Ala Ser Gln Ile Asp Ile Glu Arg Glu Thr Hi - #s Phe Asp Gly Thr Asn    #           350    - Tyr Leu Ala Val Ala Pro Gly Ile Val Ile Gl - #y Tyr Ala Arg Asn Glu    #       365    - Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Il - #e Thr Val Leu Pro Phe    #   380    - Arg Gly Asn Gln Leu Ser Leu Gly Met Gly As - #n Ala Arg Cys Met Ser    385                 3 - #90                 3 - #95                 4 -    #00    - Met Pro Leu Ser Arg Lys Asp Val Lys Trp    #               410    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 27 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: both              (D) TOPOLOGY: Not Relev - #ant    -     (ii) MOLECULE TYPE: other nucleic acid    #= "oligonucleotide"PTION: /desc    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #arthritidis) ORGANISM: Mycoplasma              (B) STRAIN: ATCC 14152    #EN231    (C) INDIVIDUAL ISOLATE:              (G) CELL TYPE: unicellu - #lar    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #             27   ATTT GACAGTA    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 33 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: both              (D) TOPOLOGY: Not Relev - #ant    -     (ii) MOLECULE TYPE: other nucleic acid    #= "oligonucleotide"PTION: /desc    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #arthritidis) ORGANISM: Mycoplasma              (B) STRAIN: ATCC 14152    #EN231    (C) INDIVIDUAL ISOLATE:              (G) CELL TYPE: unicellu - #lar organism    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #         33       CACT TAACATCTTT ACG    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: both              (D) TOPOLOGY: Not Relev - #ant    -     (ii) MOLECULE TYPE: other nucleic acid    #= "oligonucleotide"PTION: /desc    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #arthritidis) ORGANISM: Mycoplasma              (B) STRAIN: ATCC 14152    #EN231    (C) INDIVIDUAL ISOLATE:              (G) CELL TYPE: unicellu - #lar organism    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #                 22TCA CC    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: both              (D) TOPOLOGY: Not Relev - #ant    -     (ii) MOLECULE TYPE: other nucleic acid    #= "oligonucleotide"PTION: /desc    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #arthritidis) ORGANISM: Mycoplasma              (B) STRAIN: ATCC 14152    #EN231    (C) INDIVIDUAL ISOLATE:              (G) CELL TYPE: unicellu - #lar organism    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    #21                TGTT G    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 410 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: protein    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #arginini (A) ORGANISM: Mycoplasma              (B) STRAIN: LBIF              (G) CELL TYPE: unicellu - #lar organism    -      (x) PUBLICATION INFORMATION:    #al.,     (A) AUTHORS: Ohno, et    #and Immunity JOURNAL: Infection              (D) VOLUME: 58              (F) PAGES: 3788-3795              (G) DATE: November-1990    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - Met Ser Val Phe Asp Ser Lys Phe Lys Gly Il - #e His Val Tyr Ser Glu    #                 15    - Ile Gly Glu Leu Glu Ser Val Leu Val His Gl - #u Pro Gly Arg Glu Ile    #             30    - Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Le - #u Leu Phe Ser Ala Ile    #        45    - Leu Glu Ser His Asp Ala Arg Lys Glu His Ly - #s Ser Phe Val Ala Glu    #    60    - Leu Lys Ala Asn Asp Ile Asn Val Val Glu Le - #u Ile Asp Leu Val Ala    #80    - Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala Ly - #s Asp Lys Leu Ile Glu    #                95    - Glu Phe Leu Asp Asp Ser Glu Pro Val Leu Se - #r Glu Glu His Lys Val    #           110    - Val Val Arg Asn Phe Leu Lys Ala Lys Lys Th - #r Ser Arg Lys Leu Val    #       125    - Glu Ile Met Met Ala Gly Ile Thr Lys Tyr As - #p Leu Gly Ile Glu Ala    #   140    - Asp His Glu Leu Ile Val Asp Pro Met Pro As - #n Leu Tyr Phe Thr Arg    145                 1 - #50                 1 - #55                 1 -    #60    - Asp Pro Phe Ala Ser Val Gly Asn Gly Val Th - #r Ile His Tyr Met Arg    #               175    - Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe Se - #r Arg Phe Val Phe Ser    #           190    - Asn His Pro Lys Leu Ile Asn Thr Pro Trp Ty - #r Tyr Asp Pro Ser Leu    #       205    - Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Il - #e Tyr Asn Asn Asp Thr    #   220    - Leu Val Val Gly Val Ser Glu Arg Thr Asp Le - #u Gln Thr Val Thr Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Leu Ala Lys Asn Ile Val Ala Asn Lys Glu Cy - #s Glu Phe Lys Arg Ile    #               255    - Val Ala Ile Asn Val Pro Lys Trp Thr Asn Le - #u Met His Leu Asp Thr    #           270    - Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Le - #u Tyr Ser Pro Ile Ala    #       285    - Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Le - #u Val Asn Gly Gly Ala    #   300    - Glu Pro Gln Pro Val Glu Asn Gly Leu Pro Le - #u Glu Gly Leu Leu Gln    305                 3 - #10                 3 - #15                 3 -    #20    - Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pr - #o Ile Ala Gly Glu Gly    #               335    - Ala Ser Gln Met Glu Ile Glu Arg Glu Thr Hi - #s Phe Asp Gly Thr Asn    #           350    - Tyr Leu Ala Ile Arg Pro Gly Val Val Ile Gl - #y Tyr Ser Arg Asn Glu    #       365    - Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly Il - #e Lys Val Leu Pro Phe    #   380    - His Gly Asn Gln Leu Ser Leu Gly Met Gly As - #n Ala Arg Cys Met Ser    385                 3 - #90                 3 - #95                 4 -    #00    - Met Pro Leu Ser Arg Lys Asp Val Lys Trp    #               410    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 410 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: unknown    -     (ii) MOLECULE TYPE: protein    -    (iii) HYPOTHETICAL: NO    -     (vi) ORIGINAL SOURCE:    #hominis  (A) ORGANISM: Mycoplasma              (B) STRAIN: PG21              (G) CELL TYPE: unicellu - #lar    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - Met Ser Val Phe Asp Ser Lys Phe Asn Gly Il - #e His Val Tyr Ser Glu    #                15    - Ile Gly Glu Leu Glu Thr Val Leu Val His Gl - #u Pro Gly Arg Glu Ile    #            30    - Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Le - #u Leu Phe Ser Ala Ile    #        45    - Leu Glu Ser His Asp Ala Arg Lys Glu His Gl - #n Glu Phe Val Lys Ile    #    60    - Met Lys Asp Arg Gly Ile Asn Val Val Glu Le - #u Thr Asp Leu Val Ala    #80    - Glu Thr Tyr Asp Leu Ala Ser Lys Ala Ala Ly - #s Glu Glu Phe Ile Glu    #                95    - Thr Phe Leu Glu Glu Thr Val Pro Val Leu Th - #r Glu Ala Asn Lys Lys    #           110    - Ala Val Arg Ala Phe Leu Leu Ser Gln Lys Pr - #o Thr His Glu Met Val    #       125    - Glu Phe Met Met Ser Gly Ile Thr Lys Tyr Gl - #u Leu Gly Val Glu Ser    #   140    - Glu Asn Glu Leu Ile Val Asp Pro Met Pro As - #n Leu Tyr Phe Thr Arg    145                 1 - #50                 1 - #55                 1 -    #60    - Asp Pro Phe Ala Ser Val Gly Asn Gly Val Th - #r Ile His Phe Met Arg    #               175    - Tyr Ile Val Arg Arg Arg Glu Thr Leu Phe Al - #a Arg Phe Val Phe Arg    #           190    - Asn His Pro Lys Leu Val Lys Thr Pro Trp Ty - #r Tyr Asp Pro Ala Met    #       205    - Lys Met Pro Ile Glu Gly Gly Asp Val Phe Il - #e Tyr Asn Asn Glu Thr    #   220    - Leu Val Val Gly Val Ser Glu Arg Thr Asp Le - #u Asp Thr Ile Thr Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Va - #l Glu Phe Lys Arg Ile    #               255    - Val Ala Ile Asn Val Pro Lys Trp Thr Asn Le - #u Met His Leu Asp Thr    #           270    - Trp Leu Thr Met Leu Asp Lys Asn Lys Phe Le - #u Tyr Ser Pro Ile Ala    #       285    - Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Le - #u Val Asn Gly Gly Ala    #   300    - Glu Pro Gln Pro Val Leu Asn Gly Leu Pro Le - #u Asp Lys Leu Leu Ala    305                 3 - #10                 3 - #15                 3 -    #20    - Ser Ile Ile Asn Lys Glu Pro Val Leu Ile Pr - #o Ile Gly Gly Ala Gly    #               335    - Ala Thr Glu Met Glu Ile Ala Arg Glu Thr As - #n Phe Asp Gly Thr Asn    #           350    - Tyr Leu Ala Ile Lys Pro Gly Leu Val Ile Gl - #y Tyr Asp Arg Asn Glu    #       365    - Lys Thr Asn Ala Ala Leu Lys Ala Ala Gly Il - #e Thr Val Leu Pro Phe    #   380    - His Gly Asn Gln Leu Ser Leu Gly Met Gly As - #n Ala Arg Cys Met Ser    385                 3 - #90                 3 - #95                 4 -    #00    - Met Pro Leu Ser Arg Lys Asp Val Lys Trp    #               410    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 410 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: Not Relev - #ant    -     (ii) MOLECULE TYPE:    -    (iii) HYPOTHETICAL: NO    -     (iv) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:    #orale    (A) ORGANISM: Mycoplasma              (B) STRAIN: FERM BP-197 - #0              (G) CELL TYPE: unicellu - #lar    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - Met Ser Val Phe Ser Asp Lys Phe Asn Gly Il - #e His Val Tyr Ser Glu    #                15    - Ile Gly Asp Leu Glu Ser Val Leu Val His Gl - #u Pro Gly Leu Glu Ile    #            30    - Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu Le - #u Leu Phe Ser Ala Ile    #        45    - Leu Glu Ser Thr Asp Ala Arg Lys Glu His Ly - #s Glu Phe Val Glu Glu    #    60    - Leu Lys Lys Gln Gly Ile Asn Val Val Glu Le - #u Val Asp Leu Val Val    #80    - Glu Thr Tyr Asn Leu Val Asp Lys Lys Thr Gl - #n Glu Lys Leu Leu Lys    #                95    - Asp Phe Leu Asp Asp Ser Glu Pro Val Leu Se - #r Pro Glu His Arg Lys    #           110    - Ala Val Glu Lys Leu Leu Lys Ser Leu Lys Se - #r Thr Lys Glu Leu Ile    #       125    - Gln Tyr Met Met Ala Gly Ile Thr Lys Tyr As - #p Leu Gly Ile Lys Ala    #   140    - Asp Lys Glu Leu Ile Val Asp Pro Met Pro As - #n Leu Tyr Phe Thr Arg    145                 1 - #50                 1 - #55                 1 -    #60    - Asp Pro Phe Ala Ser Val Gly Asn Gly Val Th - #r Ile His Tyr Met Arg    #               175    - Tyr Lys Val Arg Asn Arg Glu Thr Leu Phe Se - #r Lys Phe Ile Phe Thr    #           190    - Asn His Pro Lys Leu Val Lys Thr Pro Trp Ty - #r Tyr Asp Pro Ala Met    #       205    - Lys Leu Ser Ile Glu Gly Gly Asp Val Phe Il - #e Tyr Asn Asn Asp Thr    #   220    - Leu Val Val Gly Val Ser Glu Arg Thr Asp Le - #u Glu Thr Ile Thr Leu    225                 2 - #30                 2 - #35                 2 -    #40    - Leu Ala Lys Asn Ile Lys Ala Asn Lys Glu Cy - #s Glu Phe Lys Arg Ile    #               255    - Val Ala Ile Asn Val Pro Lys Trp Thr Asn Le - #u Met His Leu Asp Thr    #           270    - Trp Leu Thr Met Leu Asp Lys Asp Lys Phe Le - #u Tyr Ser Pro Ile Ala    #       285    - Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp Le - #u Val Asn Gly Gly Ser    #   300    - Asn Pro Glu Pro Val Val Asn Gly Leu Pro Le - #u Asp Lys Leu Leu Glu    305                 3 - #10                 3 - #15                 3 -    #20    - Ser Ile Ile Asn Lys Lys Pro Val Leu Ile Pr - #o Ile Ala Gly Lys Gly    #               335    - Ala Thr Glu Ile Glu Thr Ala Val Glu Thr Hi - #s Phe Asp Gly Thr Asn    #           350    - Tyr Leu Ala Ile Lys Pro Gly Val Val Val Gl - #y Tyr Ser Arg Asn Val    #       365    - Lys Thr Asn Ala Ala Leu Glu Ala Asn Gly Il - #e Lys Val Leu Pro Phe    #   380    - Lys Gly Asn Gln Leu Ser Leu Gly Met Gly As - #n Ala Arg Cys Met Ser    385                 3 - #90                 3 - #95                 4 -    #00    - Met Pro Leu Ser Arg Lys Asp Val Lys Trp    #               410    __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid molecule encoding an arginine deiminase enzyme comprising an amino acid sequence of SEQ ID NO:2.
 2. The isolated nucleic acid molecule of claim 1 comprising a nucleic acid sequence of SEQ ID NO:1.
 3. An isolated nucleic acid molecule comprising a nucleic acid sequence according to SEQ ID NO:1 wherein said nucleic acid sequence is mutated by nucleotide substitutions selected from the group consisting of replacing C with T at nucleotide 39, replacing T with C at nucleotide 104, replacing G with A at nucleotide 206, replacing G with A at nucleotide 729, replacing A with G at nucleotide 337, replacing T with C at nucleotide 830, replacing T with C at nucleotide 1023, replacing T with A at nucleotide 6 replacing C with T at nucleotide 15 and replacing T with C at nucleotide 18, and combinations thereof.
 4. The isolated nucleic acid molecule of claim 3 that encodes a protein comprising the amino acid sequence of SEQ ID NO:2.
 5. The isolated nucleic acid molecule of claim 1 that is selected from the group consisting of an RNA molecule and a DNA molecule.
 6. An isolated nucleic acid molecule that is complementary to the nucleic acid molecule of claim
 1. 7. An expression vector comprising the nucleic acid molecule of claim 1 operably linked to a control sequence.
 8. The expression vector of claim 7 wherein said control sequence comprises a promoter.
 9. The expression vector of claim 8 wherein said promoter is inducible.
 10. The expression vector of claim 7 wherein said promoter is selected from the group consisting of a beta-lactamase promoter, a lac promoter, a trp promoter, a phoA promoter, an araBAD promoter, a T7 promoter, derivatives of the lambda PL and PR promotor and an OL/PR hybrid promoter.
 11. The expression vector of claim 7 selected from the group consisting of a plasmid, a phage and a cosmid.
 12. A recombinant host cell comprising the nucleic acid molecule of claim
 1. 13. The host cell of claim 12 that is prokaryotic.
 14. The host cell of claim 12 selected from the group consisting of a prokaryotic host cell and a eukaryotic host cell.
 15. The host cell of claim 12 selected from the group consisting of a bacterium and a yeast.
 16. The host cell of claim 15, that is Escherichia coli.
 17. A process for producing arginine deiminase comprising:a) culturing the host cell of claim 12 and b) expressing arginine deiminase in said host cell.
 18. A process for producing arginine deiminase comprising:a) transforming or transfecting a host cell with the vector of claim 7; b) culturing the transformed or transfected host cell; and c) expressing arginine deiminase in said cultured host cell.
 19. The process of claim 18 further comprising recovering said expressed arginine deiminase.
 20. The process of claim 19 wherein said arginine deiminase is recovered by a process comprising extracting said expressed arginine deiminase from said host cell and renaturing said expressed arginine deiminase.
 21. The process of claim 18 wherein said expressed arginine deiminase protein has a pI of 5.25.
 22. The process of claim 18 wherein said expressed arginine deiminase enzyme has serine as an N-terminal residue, wherein said N-terminal is determined following post-translational removal of methionine.
 23. The process of claim 18 wherein said expressed arginine deiminase enzyme has two cysteines per subunit.
 24. The process of claim 18 wherein said expressed arginine deiminase enzyme comprises the amino acid sequence of SEQ ID NO:2.
 25. The process of claim 18 wherein said expressed arginine deiminase enzyme has a specific activity of from about 3 to about 30 IU/mg.
 26. An isolated nucleic acid molecule that is amplified from the genome of Mycoplasma arthritidis ATCC 14152 by a polymerase chain reaction using a primer pair selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4; and said isolated nucleic acid molecule encodes an inine deiminase enzyme.
 27. The isolated nucleic acid molecule of claim 26 wherein said encoded arginine deiminase enzyme has a pI of 5.25.
 28. The isolated nucleic acid molecule of claim 26 wherein said encoded arginine deiminase enzyme has serine as an N-terminal residue, wherein said N-terminal is determined following post-translational removal of methionine.
 29. The isolated nucleic acid molecule of claim 26 wherein said encoded arginine deiminase enzyme has two cysteines per subunit.
 30. The isolated nucleic acid molecule of claim 26 wherein said encoded arginine deiminase enzyme comprises the amino acid sequence of SEQ ID NO:2.
 31. The isolated nucleic acid molecule of claim 26 wherein said encoded arginine deiminase enzyme has a specific activity of from about 3 to about 30 IU/mg.
 32. The isolated nucleic acid molecule of claim 26 wherein SEQ ID NO:3 is modified at base 6 so that A is substituted for T; at base 15 so that T is substituted for C; and at base 18 so that C is substituted for T, and combinations thereof.
 33. The isolated nucleic acid molecule of claim 26 that is selected from the group consisting of an RNA molecule and a DNA molecule.
 34. The host cell of claim 26 that is a bacterium. 